Rubber-like material for the immobilization of proteins and its use in lighting diagnosis and biocatalysis

A rubber-like, protein-based technology used in biochemical equipment and methods, microbial assay/inspection, luminescent materials, etc.

Inactive Publication Date: 2018-05-11
FUNDACION IMDEA MATERIALES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The present invention addresses the above-discussed shortcomings and solves the problem of providing new and improved methods of immobilizing and stabilizing a wide range of different proteins, including photoproteins and enzymes, without any need for cross-linking or immobilization

Method used

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  • Rubber-like material for the immobilization of proteins and its use in lighting diagnosis and biocatalysis
  • Rubber-like material for the immobilization of proteins and its use in lighting diagnosis and biocatalysis
  • Rubber-like material for the immobilization of proteins and its use in lighting diagnosis and biocatalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0341] Example 1 : Preparation of Rubber-like Materials Containing Immobilized Proteins

[0342] Preparation of proteins and enzymes (expression and purification of recombinant proteins)

[0343] Such as figure 1As shown, several different photoproteins and enzymes were prepared and characterized. Escherichia coli (E.coli) strain M15[pREP4] carrying the appropriate plasmid (both pQE-9 expression constructs containing the N-terminal 6xHis tag from the pQE-9 expression vector, Qiagen) was incubated at 28°C in the presence of Amp (200 μg / ml) and Kan (100 μg / ml) antibiotics grown in LB medium to an optical density at 600 nm of about 0.5. Recombinant protein expression was induced using 1 mM isopropyl-β-D-1-thiogalactoside at 28°C. After 4 hours of growth at 28°C, cells were harvested and frozen at -20°C. Frozen bacterial cells were thawed and lysed chemically using lysozyme and mechanically using a sonicator. Then, following the QIAGEN protocol (Henco K, A handbook for hi...

Embodiment 2

[0357] Example 2 : Preparation of rubber-like materials using different mass ratios of branched polymers and linear polymers and different amounts of buffered aqueous solutions

[0358] The preparation of gels and rubber-like materials according to the present invention uses different mass ratios of branched polymers (in this example, M n 450 Da ethoxylated trimethylolpropane (TMPE)) and linear polymers (in this example, M n 5000 kDa polyethylene oxide (PEO)), as shown in Table 1 and Table 2 below. Rubber formation was performed as described in Example 1.

[0359] Specifically, the above two polymers were mixed in different mass ratios as shown in Table 1 and Table 2 below. Although TMPE is a low-viscosity liquid, PEO does not dissolve even under high-speed stirring conditions. To facilitate this process, different amounts of buffer (again for proteins) were added.

[0360] Table 1:

[0361]

[0362] Table 2:

[0363]

[0364]As summarized in Table 1, until 200 µ...

Embodiment 3

[0369] Example 3: Applications of rubber-like materials containing immobilized proteins as downconversion encapsulation systems

[0370] Figure 5 A schematic diagram showing a hybrid light-emitting diode with a rubber-like material containing immobilized proteins therein as a down-converting packaging system (see Example 1). A commercially available blue emitting LED with an electroluminescence spectrum at 450 nm (from Luxeon) was used in this example. To coat the previous silicone encapsulation in three-dimensional form, the LED can either be immersed in the gel for a few seconds and / or the gel can be deposited by drop coating onto the surface of the carrier. Subsequently, the coating was dried as described in Example 1 above. Devices were driven under constant and / or pulsed current and voltage regimes. In this example, a Keithley 2400 was used to drive the LED at a constant current of 10 mA, and the electroluminescence spectrum and device performance was monitored usin...

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Abstract

The present invention relates to a process of preparing a rubber-like material containing a protein immobilized therein as well as a corresponding rubber-like material the process comprising the stepsof (a) mixing a protein a branched polymer such as trimethylolpropane ethoxylate and a linear polymer such as poly( ethylene oxide) in an aqueous solution to form a gel and (b) drying the gel to obtain a rubber-like material containing the protein immobilized therein wherein the branched polymer comprises at least three polymeric branches bound to a central branching unit. The rubber-like material allows the immobilization and stabilization of a wide range of different proteins including luminescent proteins as well as enzymes and can particularly advantageously be used as down-converting material for light-emitting diodes (LEDs) for diagnostic applications and in bioreactors.

Description

technical field [0001] The present invention relates to a method for the preparation of rubbery materials containing immobilized proteins therein, and corresponding rubbery materials, said method comprising the steps of: (a) mixing proteins, such as ethoxylated trimethylol A branched chain polymer of propane and a linear polymer such as polyethylene oxide form a gel, and (b) drying the gel to obtain a rubbery material containing immobilized proteins therein, wherein the branched chain polymer Comprising at least three polymeric branches bonded to a central branching unit. This rubbery material can immobilize and stabilize a wide range of different proteins, including photoproteins and enzymes, and is particularly advantageous as a down-converting material for light-emitting diodes (LEDs) for diagnostic applications and in the bioreactor. Background technique [0002] Protein immobilization for biotechnological processes has been considered the most cost-effective means of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): H01L33/50C07K17/02
CPCB82Y5/00C08G2650/30C09D171/02C07K17/04C09K11/025C09K11/06C12N9/1205C12N9/2431C12N9/92C12N9/96C12Y207/01001C12Y503/01009C08G65/333C07K17/08C08J3/075C08J2371/02C08J2471/02C08J2489/00C09K2211/14C12Q1/008C12Q1/40C12Q1/485C12Q1/533H01L33/56
Inventor R·D·科斯塔,瑞克末U·索尼瓦拉德P·布拉纳,科拓M·D·瓦贝M·普拉塞尔
Owner FUNDACION IMDEA MATERIALES
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